Drug resistance in Plasmodium falciparum (Pf), the deadliest of the malaria parasites that threatens almost half the world?s population, has been associated with genetic changes in specific parasite alleles from field isolates. The protein responsible for Pf asexual blood stage (ABS) parasite resistance to both previously and currently used first-line antimalarials, chloroquine (CQ) piperaquine (PPQ) and amodiaquine (ADQ), is the 48-kDa P. falciparum chloroquine resistance transporter (PfCRT). CQ, PPQ and ADQ, all 4-aminoquinolines, eliminate drug-sensitive Pf ABS parasites by inhibiting the detoxification of host heme, a product of parasite-mediated hemoglobin degradation, inside their digestive vacuole (DV). PfCRT, situated in the DV membrane, is thought to mediate CQ resistance via drug efflux. Our progress in understanding how PfCRT functions, and the molecular basis of PfCRT-mediated drug resistance, has been seriously hampered by the lack of an atomic model of this transporter. Using antigen-binding fragment (Fab) technology and single-particle cryo-electron microscopy, we have determined the structure of the full-length CQ-resistant 7G8 mutant isoform of this 10- transmembrane protein to 3.2 resolution, in an inward-open conformation. These preliminary data are presented herein, together with functional assays using purified protein in nanodiscs and in liposomes, and parasite-based assays with pfcrt-modified lines. In this application, we propose to compressively define PfCRT structure and function and leverage this into experimentally testable predictions of how PfCRT can further evolve to drive new patterns of multidrug resistance across malaria-endemic regions. In Aim 1, we will solve the PfCRT structure for globally variant isoforms, including complexes with the antimalarial drugs CQ, PPQ and ADQ, and physiologic substrates. In Aim 2, we will implement biophysical approaches with recombinant protein to elucidate the natural function of PfCRT and develop a model of PfCRT-mediated substrate and drug transport. In Aim 3, we will apply validated gene-editing approaches to predict emerging PfCRT-mediated resistance and elucidate its functional impact in parasites. Gene-editing studies will focus on PPQ and ADQ in the context of major global PfCRT variants, as a way to anticipate how mutant PfCRT could evolve new drug resistance traits, including with isoforms present in high-transmission African settings where drug-resistant malaria exerts by far its greatest impact. This coordinated research effort combines three Columbia University groups led by Drs. Mancia, Quick and Fidock, who bring expertise in membrane protein biochemistry and structure, bioenergetics of membrane transport, and Pf biology including mechanisms of antimalarial drug resistance, respectively. This highly integrated project has the potential to transform our understanding of how PfCRT mediates multidrug resistance, by providing powerful advances in deciphering PfCRT structure and function, delivering new biomarkers of emerging resistance, and identifying antimalarial combinations that could be used regionally to effectively treat drug-resistant Pf malaria.